肝機能障害の評価法とその障害機序に関する研究; ヒト肝 Title ... ·...

Title

肝機能障害の評価法とその障害機序に関する研究; ヒト肝ミトコンドリアの日内代謝変動に基づく肝機能評価及び動物モデルを用いた肝ミトコンドリア障害機序(Dissertation_全文 )

Author(s) Iwata, Shingo

Citation Kyoto University (京都大学)

Issue Date 1993-01-23

URL https://doi.org/10.11501/3091221

Right 主論文は出版社の許諾条件により公開していない部分があります

Type Thesis or Dissertation

Textversion author

Kyoto University

m

~.m 13 Jjf ~ fiE ~ ~ (J) ~ {iHj ~ t. -f (J) ~ ~ 11 F¥ ~= ~ 9 Q "6Jf ~ ; 1::: I' llf ~ I' ~ / j-: 'J 7 (J) B !1'1 ft ~ ~ t1J 1:: ~ -:5 < Jlf 11 fiE ~ fi1li

& u tJJ ~ ~ -r Jv ~ m v' t::. llf ~ " ~ / j-: 'J 7 fSl ~ mt F¥

1 1

[

' ,A, " :r:r-@)2-GASTROENTEROLOGY 1991 :100:1371-1378

Diurnal Fluctuations of Arterial Ketone Body Ratio in Normal Subjects and Patients With Liver Dysfunction

SHINGO IWATA, KAZUE OZAWA, YASUYUKI SHIMAHARA. KEIICHIRO MORI, NOBUAKJ KOBAYASHI. KAORU KUMADA, and YOSHIO YAMAOKA Second Department of Surgery. Faculty o f Medicine. Kyoto University. Kvoto. Japan

To explore the metabolic aspects of chronic liver disease, diurnal changes of arterial ketone body ratio (acetoacetate/3-hydroxybutyrate), reflecting he· patic mitochondrial redox potential, were investi· gated in normal subjects, patients with chronic liver disease (Child's class A or 8), and patients with hepatic failure (Child's class C). Ketone body ratio in normal subjects increased after breakfast from 0.96 to 2.00, after lunch from 2.17 to 2.38, and after dinner from 1.23 to 2.55 with blood glucose level ranging from 103 to 141 mg/dL (5.7 to 7.8 mmoVL). By contrast, the ketone body ratio in the Child A orB group changed little and remained within a range of 0. 7o-1.35 despite a large change in blood glucose level from 102 to 176 mg/dL (5 . 7 to 9.8 mmoVL). Ketone body ratio in Child's class C remained near or below 0.4 with no response to glucose administra· tion, despite a marked elevation in blood glucose level. These results indicate that hepatic mitochon· drial redox potential undergoes diurnal changes in sharp response to meals in normal liver function but that these fluctuations are absent in patients with liver damage (Child's class A, 8 , and C). Further· more, it remains at low levels in severe liver fai lure (Child's class C). It is also suggested that hepatic mitochondrial redox potential plays an important role in the regulation of blood glucose levels.

Since we proposed the redox theory in the early 1980s (1,2), our research has been focused on the

essential role of arterial ketone body ratio (AKBR; aceloacetale/3-hydroxybutyrate) in energy metabo· !ism as lhe metabolic basis of multiple systemic organ failure (3,4). ExperimentaJ findings indicate that AKBR is an accurate parameter to estimate the hepatic function in primary liver disease as well as in hepatectomy, hypoxia. hemodilution. and hemorrhagic shock

(5-9). The redox theory is now receiving wider acceptance. and AKBR has begun to be applied clinically to evaluate the changes in hepatic function (10-13). More recently, AKBR has been reported to have potential use as an early indicator of graft failure after human liver transplantation (1 4,15) .

The AKBR decreases in patients with liver disease, and it remains below 0.4 in patients with severe hepatic failure, at which level it hardly responds to any form of intensive treatment. Eighty-five percent of the patients with AKBR below 0.4 die of liver fa ilure complicated by multiple organ insufficiency (1,2.1 l ). On the other hand. the AKBR in the normal liver rapidly responds to factors such as hepatic blood flow. glucose load, and insulin and attains high levels over 0.7 (16.17).

This study aims to clarify the extent of fluctuation in AKBR in the cycle of daily life. The diurnal changes in AKBR were measured in normal subjects. patients with chronic liver disease, and patients with severe hepatic failure. Our findings show that a different pattern in diurnal changes of AKBR exists between normal and impaired livers and that diurnal changes in AKBR have a tendency to flatten and remain at low levels in accordance with the severity of liver dysfunction.

Materials and Methods

This stud~· was performed in six young male volunteers as a control group (group 1). seven palients with c hronic liver disease (group 2). and three patients wtth severe hepatic failure (group 3) under informed consent

Abbreviations used in this paper: AKBR. arterial ketone body ratio: TCA. tricarboxylic a cid .

' 1991 by the American Gastroenterological Association 0016·5085/91/SJ.OO

Alterations in the proton J\TPase activity of rat-liver mitochondria after hemorrhagic shock

SHINGO IWATA, AKIRA TANAKA, and KAZUE OZAWA

From the Second Department of Surgery, Faculty of Medicine, Kyoto University, l<yoto, Japan

This study was supported in part by grants from the Scientific Research Fund of the Ministry of Education and by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan.

Address for reprint requests and correspondence to: Shingo Iwata M.D., Second Department of Surgery, Faculty of Medicine, Kyoto University, 54, Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan. Business phone:81-75-751-3666, Fax:8:1-75-753-2908, Home phone:81-75-256-0874

1

ABSTRACT

To clarify the damage s ite of compl icated oxidative phosphorylation

function after hemorrhagic shock in jaundiced liver mitochondria,

the H+-ATPase activity of inside--out submitochondrial particles,

mitochondrial membrane potential, and oxygen consumption in the

presence of uncoupler were studied as indices of phosphorylation,

membrane intactness, and oxidation, respectively . Hemorrhagic shock

was induced according to the Wig~Jers' model (mean arterial blood

pressure=40 mmHg) in rats made jo1undiced by common bile duct

ligation; sham-operated rats served as controls. After reinfusion

of the shed blood, all of the control rats survived but all of the

jaundiced rats died. Liver mitochondria from jaundiced rats after 1

hr hypotension demonstrated a 48% decrease in mitochondrial ATPase

activity without remarkable chang,es in either oxidative activity or

membrane potential of liver mitochondria. The reduction of ATPase

activity appeared to be due to its release 1n the supernatants

obtained from submitochondrial particles since the ATP activity of

supernatants in jaundiced rats was significantly (p<0.001) higher

than that of the controls. It is suggested that this enzyme plays a

key role in energy restoration in recovery from shock.

2

Running head: Proton ATPase ln shock

Abbreviations: ADP = adenosine diphosphate; ATP = adenosine

triphosphate; ESMP = submitochondrial particles prepared in the

presence of ethylenediaminetetraacetic acid; F0 = membrane integral

sector of mitochondrial proton adenosinetriphosphatase complex; F1 =

catalytic part of mitochondrial proton adenosinetriphosphatase

complex; FCCP = carbonyl cyanide p-trifluoromethoxyphenylhydrazone;

H+-ATPase = proton adenosinetriphosphatase complex. SDS = sodium

dodecyl sulfate; PAGE = polyacrylamide gel electrophoresis

3

INTRODUCTION

Many clinical studies have demonstrated that opertation on severe

obstructive jaundice is associated with a postope1rative morbidity

of 40-60% and a mortality of 15-25 %.1-6 Postoperative

complications and causes of death include renal and hepatic

failure, infection and hemorrhage. Bile duct ligation induces many

biological alterations including hemodynamic and n1etabolic

abnormalities.?-10 Once metabolic functions of the liver are

impaired with biliary obstruction, the impairment takes six weeks

or more to improve after release of obstruction.10 Experimental

animals with obstructive jaundice have an increased susceptibility

to hemorrhagic hypotension7,8 and have an increased tendency to

develop shock following hemorrhage.u Although som•e studies deal

with the hemodynamic abnormalities in these animals, the metabolic

aspects of obstructive jaundiced liver during hemorrhagic shock

have not been well documented. Previous reports12,13 from our

laboratory showed that hemorrhagic shock for two hours in

obstructive jaundiced animals proved lethal and that severe

impairment of mitochondrial oxidative phosphorylati[on was

observed. The authors thought that poor recovery of energy state

4

after reinfusion of the reserved blood due to the impairment of

oxidative phosphorylation may be reloted to so-called irreversible

shock. It has been reported that the decline in ATP levels or

energy state is a critical event in the development of cell

damage.14,1S Although it may be controversial whether abnormalities

in ATP levels and ATP production have relevance for the high

mortality rate, low ATP levels at least lead to inactivation of

energy-requiring systems. Maintenance of ATP levels or ATP

production systems is thought be important for critical cellular

functions .16,17

Oxidative phosphorylation should be functionally and

structurally separable into oxidative proton translocating and

phosphorylating proton translocating complexes, namely electron

transport proteins and H+-ATPase, respectively.18 These two systems

are coupled to produce ATP by means of an electrochemical proton

gradient.19 In this study, in order to clarify the damage site of

complicated oxidative phosphorylation system using obstructive

jaundiced rats subjected to hemorrhagic shock, the H+-ATPase

activity of inside-out submitochondrial particles and oxygen

consumption of mitochondria in the presence of an uncoupler were

5

studied as indices of phosphorylation and oxidation, respectively.

It is also important to determine whether the mito•chondrial

membrane is intact since membrane permeability greatly affects

oxidative phosphorylation. Therefore the mitochondl~ial membrane

potential was measured as an indicator of membrane intactness.

Evidence will be presented indicating that the disability of

phosphorylation occurred in jaundiced liver mitochondria at 1 hr

after hemorrhagic shock. H+-ATPase is comprised of the membrane

sector (F0 ) and the catalytic part CF1). The F1 consists of the

five subunits (a, p, y, b , and E in order of decreasing molecular

weight). It is known that only the a and p subunits bind ATP and

that p subunit is a catalytic center of ATPase. Molecular changes

in H+-ATPase were also studied by oligomycin sensitivity of ATPase

and SDS-PAGE analysis of submitochondrial particles.

METHODS

Male albino rats of Wistar strain were maintained on Cleo 2

(Nippon Haigoshiryo Co Ltd., Osaka, Japan) and water ad libitum

for two weeks before the operation. Those weighing (Jbout 150 grams

6

constant mean arterial blood pressure. 20 After 1 or 2 hr

hypotension, all reserved blood was returned to the rats . At 24

hr after the induction of shock, the survival rate was 0% (0/10)

1n jaundiced rats, but 100% (10/10) in control rats. All

procedures were performed under conditions as sterile as possible.

All experiments involving animals were carried out 1n accordance

with the institution's guidelines for the care and use of

laboratory animals.

Liver mitochondria were prepared after 1 or 2 hr hypotension by

the method described previously.Zl

Oxidative phosphorylation function was monitored

polarographically by the conventional method previously reported.ZZ

Oxidative activity was evaluated polarographicallly by measuring

oxygen consumption rate in the presence of FCCP as an uncoupler.

Assay for oxidative activity was done at 22°( (pH ?.4) in a medium

containing 0.3 M mannitol, 10 mM potassium chloride, 2 mM magnesium

chloride, 10 mM tris(hydroxymethyl)aminornethane-hyalrochloride

buffer, 3 mM potassium phosphate buffer, and 0.2 m~l

ethylenediaminetetraacetic acid. Glutamate as a substrate was added

at a concentration of 3 mM and FCCP was added at concentration of 1

8

~M. The oxygen consumption rate i.n the presence of FCCP was

calculated from the polarographic tracings.

Mitochondrial membrane potentic1l was measured by using FCCP

according to the method of Akermcm23 which was modified as follows.

The reaction medium contained 0.2 M mannitol 0.38 mM

ethylenediaminetetraacetic acid, 20 mM

N-2-hydroxyethyl -piperazine-N'-2-ethane sulfonic acid (pH 7.4 with a

tris(hydroxymethyl)aminomethane buffer) and 20 mM potassium chloride

in 3 ml final volume. Safranine 0 was used in a final concentration

of 9.6 ~M. The reactions were sttarted by addition of freshly

isolated mitochondria to reaction medium containing safranine 0,

wi.th (state 4) or without (state l) addition of glutamate as

substrate. When absorbance change reached equilibrium, 0.48 ~M FCCP

was added, which resulted in a rapid absorbance change. Reactions

were performed in cuvettes of 1cm light path at 22°(. Changes 1n

absorption spectra were recorded vrith a two-wave length double beam

spectrophotometer (UV 3000, Shimadzu Co Ltd., Kyoto, Japan) set at

511 to 533 nm. All measurements were done in triplicate. The

membrane potential was calibrated by the potassium diffusion

potential. Valinomycin induced potassium diffusion potential was

9

calculated from the Nernst equation us1ng values of [K+]i. and [K+] 0

in the presence of valinomycin: dE=60 X log[K+]i./[K+] 0 where dE is

the membrane potential in millivolts, and [K+] i. and [K+] 0 represent

the internal (matrix) and external (medium) concentrations of

potassium ion, respectively, with [K+]i. assumed to be 120 mM .23

Preparation of ESMP and determination of ATPase activity were

performed as followed . Purified submitochondrial particles were

prepared by a procedure modified from that of Lee et al . . 24 Freshly

isolated mitochondria were stored for 7 days at -30°( and thawed

immediately before use. They were suspended at a concentration of

20 mg protein/ ml in 0.3 M mannitol, containing 2 mM

ethylenediamine-tetraacetic acid (final pH 8.5), and were then

exposed to ultrasonic energy for 60 s at 4°( (Heat Systems

Ultrasonics , Model W-225, output 70 W). Large mitochondrial

fragments were removed by centrifugation at 10000g for 10 min.

Submitochondrial particles were sedimented from the resulting

supernatant at 100000 g for 40 min at 4°( . The sediment was

resuspended to a final concentration of 30 mg/ ml with 0. 3 M

mannitol . ATPase activity was determined spectrophotometrically at

340 nm in a thermostatically controlled reaction cuvette at 30°( by

10

coupling the production of ADP to the oxidation of reduced

nicotinamide adenine dinucleotide v1a the pyruvate kinase and

lactate dehydrogenase reactions by a modified method of Buckle et

al .. Zs The reaction mixture contained 0.3 M mannitol, 50 mM

potassium chloride, 5 mM magnesium chloride, 20 mM

tris(hydroxymethyl)-aminomethane hydrochloride pH 7.4, 0.5 ~g/ml

rotenone, 0.1 mM reduced form of nicotinamide adenine dinucleotide,

1 mM phosphoenolpyruvate, 5 units/ml lactate dehydrogenase, 2

units/ml pyruvate kinase and 20-30 ~g/ml submitochondrial particles

in a final volume of 3 ml. The reaction was started by the addition

of ATP at the concentrations of 0.025 to 1.6 mM. The data were

analyzed based on the Michaelis-Menten equation to give Vmax and Km.

The oligomycin sensitivity of the ATPase activity was measured over

the range from 1.0 to 20 nmol of oligomycin.

SDS-PAGE was carried out on 12.5% polyacrylamide gels in the SDS

and 3.2 mM urea.26 Western blotting and subsequent immunodecoration

were performed as described prev·iously utilizing an anti-bovine

heart mitochondrial F1 antibody.27 Quantitative measurements of F1

subunits from Western blots were performed using Soft Lase r Scanning

Densitometer (Model SLR 20/10 Bic~ed Instrument, Inc., Fullerton,

1 1

CA, U.S.A.). Protein concentrations were measured by the method of

Lowry et al. using bovine serwm albumin as standard.28

Safranine 0 was obtained from Chroma Gesellschaft Schmid & Co.,

FCCP from Fluka A.G., phosphoenolpyruvate, nicotinamide adenine

dinucleotide reduced form, val ~nomycin and oligomycin from Sigma

Chemical Co., lactate dehydrogenase and pyruvate kinase from

Boehringer (Mannheim). Anti-bovi.ne heart mitochondrial F1 antibody

was generous gift from Dr. Yasuo Kagawa of Jichi Medical School,

Tochigi, Japan. Other reagents ~~re of the highest grade

commercially available.

All results are expressed as mean ± SEM. Statistical analysis was

made with analysis of variance and Student's t-test. Statistical

significance was obtained when the P value was less than 0.05.

RESULTS

Table 1 shows the respiratory control ratio, ADP/0 ratio, state

3 respiratory rate, and phosphor~tlation rate after induction of

hemorrhagic shock in control and jaundiced rats. The respiratory

control ratio with glutamate or succinate as substrate

significantly decreased after 1 and 2 hr hypotension 1n jaundiced

1 2

rats. Coupling of oxidation and phosphorylation is judged by

ADP/0 ratio and ADP/0 ratio did not decrease after 1 or 2 hr

hypotension in controls. By contrast, in jaundi ced rats, it did

not decrease after 1 hr hypotension but decreased significantly

after 2 hr hypotension. The state 3 respirator)' rate (n mole of

atom oxygen per m1n per mg of mitochondrial protein) and the

phosphorylation rate (n mole of ATP synthesized per min per mg of

mitochondrial protein) wi.th glutamate or succinate as substrate

decreased significantly after 1 and 2 hr hypotension in jaundiced

liver mitochondria. From the above results, the oxidative

phosphorylation activity decreased significantly after hemorrhagic

shock in jaundiced rats.

Table 2 shows the changes 1n respiration robe of liver

mitochondria in the presence of FCCP after induction of

hemorrhagic shock in control and jaundiced rats .. The

FCCP-stimulated respiration rate with glutamate as substrate tends

to decrease at 2 hr after induction of shock in jaundiced rats but

has no significant decrease. The uncoupler stimulated respiration

rate wi.th succinate has no variations even after i.nducti.on of

shock i.n control and jaundiced animals. These observations i.mply

1 3

that no remarkable damage occurs to the electron transport system.

Table 3 shows the mitochondrial membrane potential after

induction of hemorrhagic shock in control and jaundiced rats.

Membrane potential in state 1 and state 4 had no decrease during

hemorrhagic shock in jaundiced and control rats. This result

indicates that the mitochondrial membrane was intact during the

observation period.

Table 4 shows H+-ATPase activity after induction of hemorrhagic

shock in control and jaundiced rats. There was no significant

difference in the mean values of Vrrtax and Km from ESMP between

control and jaundiced rats before shock and their values were

consistent with those of a previous. report.zs By contrast,

H+-ATPase activity was drastically lowered by 48% in ESMP from

jaundiced rats after 1 hr hypotension, and by 72% after 2 hr

hypotension. These results indicate that phosphorylative activity

decreases after shock in jaundiced tanimals. Km increased 1n

jaundiced rats after shock.

SDS-PAGE was performed to exam1ne the molecular changes in five

subunits of F1. As shown in Figure l, SDS-PAGE followed by Western

blotting with anti-bovine heart mitochondrial F1 antibody indicated

14

that there was no change in the a/~ ratio 1n pur1e F1, control and

jaundiced ESMP after 1 hr hypotension. Densitometrical data of a/~

ratio were 1.01, 0.98, and 1.00, in purified F1, control, and

jaundiced ESMP, respectively. This indicates that: we can not

account for the reduction of ATPase activity observed in jaundiced

mitochondria after shock by dissociation of a or ~ subunit from

F1. Because y, o and E subunits are small proteins which are not as

easily detected as a and ~ subunits, we have no information

concerning changes of these subunits.

We examined oligomycin sensitivity and H+-ATPc1se activity of

supernatants obtained from ESMP. F0 of H+-ATPase is a

water-insoluble component that spans the energy tr·ansducing

membrane and directs protons toward or from F1 , a water-soluble

catalytic component. It is known that H+-ATPase activity is

inhibited by oligomycin and also that oligomycin sensitivity 1s

diagnostic of a correct assembly of F1 to F0 .18 As shown in Table

5, oligomycin sensitivity was significantly lowered in ESMP from

jaundiced rats after shock. ATPase activity of supc~rnatants 1n

jaundiced rats after shock was significantly higher than that of

1 5

the controls. This suggests that a significant portion of the F1

was solubilized from jaundiced ~nitochondria after shock. On the

other hand, only a small portion of F1 was released from control

mitochondria.

DISCUSSION

The present study demonstrates that oxidative phosphorylation

1s more significantly impaired in jaundiced rats subjected to

hemorrhagic shock than in controls and that this is to be due to a

defect in the terminal phosphorylation phase of oxidative

phosphorylation rather than a defect in the electron transport

chain. As an important control, vve show no change in liver

mitochondrial membrane potential suggesting that the mitochondrial

membrane remained intact during the period of hemorrhagic shock.

H+-ATPase is more vulnerable to the combined stresses of

obstructive jaundice and hemorrhagic shock, than respiratory chain

and mitochondrial membrane. A recent paper has indicated that the

membrane potential is maintained for a comparative long period

during mitochondrial injury and breaks down rather promptly beyond

1 15

a critical point.29 An other pe~per reported that ATPase activity

decreased most rapidly compared with the activities of

mitochondrial inner membrane enzyme complexes of electron

transport in rat ischemic heart.~ Experimental results obtained

from our model are compatible wi.th these papers.

The reduction of ATPase activity appears to be due to

considerable release of F1 into the supernatant. The difference tn

oligomycin sensitivity between control and jaundiced mitochondria

after shock indicates that there may be altered assembly of F1 to

F0 which interacts with oligomycin.18 Our observation demonstrates

that the water-soluble catalytic subunits, Ft, are not as tightly

bound to the inner membrane as in control mitochondria. This loss

in affinity suggests the possibility that the function of proteins

thought to make structural links between Ft and F0 has been

compromised in jaundiced mitochondria after hemorrhagic shock. The

properties of the Ft portion may be changed by perturbation of

the membrane sector subunits, causing the impairment of

phosphorylation, as supported by other investigators.31,32

It has been reported that an intrinsic inhibitor protein which

binds to the H+-ATPase exists in mitochondria.33,~ If the

1 7

inhibitor protein increases after hemorrhagic shock, especially in

jaundiced liver, H+-ATPase activity may decrease. However, it is

unlikely that the inhibitor protein can increase sufficiently to

cause an effect after 1 or Z hr of shock, especially only in

jaundiced liver. Furthermore, if the inhibitor protein dissociates

from ATPase, ATPase activity increases. But this study indicates a

decrease of ATPase activity in jaundiced rats after shock.

Therefore, there is little possibility in this case that the

inhibitor protein is important in H+-ATPase activity.

Common bile duct ligation induces alterations in many

biological phenomena, especially the elevation of bilirubin and

bile acid levels which have cytotoxic effects. Since it is known

that bile acids have an especially high hepatotoxic potential as

detergents,35,3G these bile acids may be effective in dissociation

F1 from F0 . When ATP production decreases due to disability of

phosphorylation, intracellular calcium increases because the Ca

pump 1s energy-dependent. Calciurr1 enhances the detergent activity

of bile salts3s and the decrease of ATPase activity may be

accelerated more.

Bilirubin has been shown to have an inhibitory effect on

1 8

membrane- requiring mitochondrial reactions .37 More recent study

suggests that the binding site of an organic anion such as

bilirubin is present on ~ subunit of F1.38 Bilirubin is known to

uncouple oxidative phosphorylation in mitochondria .39 This study

shows no changes before shock and after 1 hr hypotension in ADP/ 0

ratio which is an index in judging coupling of oxidation and

phosphorylation . Therefore, there 1may be little effect of

bilirubin as a uncoupler by the period of at least first 1 hr

hypotension. Tissue and albumin canpete for binding the body's

bilirubin pool. It has been reported that the binding of bilirubin

to mitochondria is not determined by the free bilirubin level, but

by the concentration of the pH dependent subfraction, the free

bilirubin acid salt.40 Since the uptake of bilirubin by

mitochondria increases at lower pH and since acidosis is profound

in the cell and the extracellular fluid in hemorrhagic shock, the

cytotoxicity of bilirubin will 1ncrease during hemorrhagic shock.

In conclusion , the activity of H+-A-rPase decreases significantly

by hemorrhagic shock in jaundiced rats without remarkable changes

1n either the oxidative activity or the membrane potential of

liver mitochondria. This decrease of activity may be explained by

19

the possibility that the bindin,g F1 to Fo is compromised. It is

suggested that the F0 F1-ATPase complex plays key role in energy

restoration in recovery from shock.

Personal acknowledgment

We thank Dr. Yasuo Kagawa of Jichi Medical School for providing

precious antibody used in this study.

20

References

1. Denning DA, Ellison EC, Carey LC. Preoperative percutaneous

transhepatic decompression lowers operative morbidity in

patients with obstructive jaundice. Am J Surg 1981;141:61-5.

2. Gundry SR, Strdel We, Knol JA, et al. Efficancy of preoperative

biliary tract decompression in patients with obstructive

jaundice. Arch Surg 1984;119:703-8.

3. Pitt HA, Gomes AS, Lois JF, Mann LL, Deutsch LS, Longmire,Jr,

WP. Does preoperative percutaneous biliary drainage reduce

operative risk or increase hospital cost? Ann Surg 1985;201:545-

53.

4. Pitt HA, Cameron Jl, Postier RG, Gadacz TR. factors affecting

mortality in biliary tract surgery. Am J Surg 1981;141:66-72.

5. Dixon JM, Armstrong CP, Duffy SW, Davies GC. Factors affecting

morbidity and mortality after surgery for obstructive jaundice:

a review of 373 patients. Gut 1983;24:845-52.

6. Blarney SL, Fearon KCH, Gilmour WH, Osborne DH, Carter DC.

Prediction of risk in biliary surgery. Br J Surg 1983;10:535-8.

7. Gali D, Blendis LM, Bomzon A. Vascular reactivity in reversible

experimental obstructive jaundice. J Surg Res 1987;42:242-6.

21

8. Green J, Beyar R, Sideman S, et al. The Jaundiced heart: A

possible explanation for postoperative shock in obstructive

jaundice. Surg 1986;100:14-9.

9. Johnstone JMS, Lee EG. A quantative assessment of the structual

changes in the rat's liver following obstruction of the common

bile duct. Br J exp path 1976;57:85-94.

10. Koyama K, Takagi Y, Experimental and clinical studies on the

effect of biliary drainage in obstructive jaundice. Am J Surg

1981;142:293-9.

11. Finberg JPM, Seidman R, Better OS. Cardiovascular

responslveness to vasoactive agents in rats with obstructive

jaundice. Clin Exp Pharmacol Physiol 1982;9:639-43.

12. Yamamoto M, Sato M, Ida T, Ukikusa M, Ozawa K. Obstructive

jaundice and hemorrhagic shock. Circ Shock 1978;5:235-49.

13. Yamamoto M, Ozawa K, Tobe T, Isselhard W. Effect of hypovolemic

hypotension on plasma proteins and hepatic energy status in

jaundiced rabbits. Eur Surg Res 1982;14:45-55.

14. Lanir A, Jenkins RL, Caldwell, et al. Hepatic transplantation

survival: correlation with adenine nucleotide level in donor

liver. Hepatology 1988;8:471-75.

22

15. Chapman AG, Fall L, Atkinson DE. Adenylate energy charge in

Escherichia coli during growth and starvation. J Bacterial

1971;108:1072-86.

16. Watanabe F, Kamiike W, Nishimura T et al. Decrease in

mitochondrial levels of adenine nucleotides and concomitant

mitochondrial dysfunction in ischemic rat liver. J Biochem

1983;94:493-9.

17. Farber JL, Chien KR, Mittnacht S Jr. The patogenesis of

irreversible cell injury in ischemia. Am J Patho 1981;102:271-

81.

18. Nicholls DG. Bioenergetics. London: Academic Press, 1982.

19. Mitchell P, Moyle J. Stoichiometry of proton translocation

through the respiratory chain and triphosphatase systems of

rat liver mitochondria. Nature 1965;208:147-51.

20. Werle JM, Cosby RS, Wiggers CJ. Observations on hemorrhagic

hypotension and hemorrhagic shock. Am J Physiol

1942; 136:401-;~0.

21. Ozawa K, Takasan H, Mizukami T. Human liver mitochondria. Clin

Chim Acta 1972;38:385-93.

22. Ozawa K, Takasan H, Kitamura 0. Effect of ligation of portal

23

ve1n on liver mitochondrial metabolism. J Biochem

1971;70:755-64.

23. Akerman KEO, Wikstrom MFK. Safranine as a probe of the

mitochondrial membrane potential. FEBS Lett 1976;68:191-7.

24. Lee CP, Ernster L. Studies of the energy-transfer system of

submitochondrial particles-- 2. Effects of oligomycin and

aurovertin. Eur J Biochem 1968;3:391-400.

25. Buckle M, Guerrieri F, Papa S. Changes in activity and F1

content of mitochondrial H+-ATPase in regenerating rat liver.

FEBS Lett 1985;188:345-51.

26. Laemmli UK. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature

1970;227:680-S.

27. Towbin H, Staehelin T, Gorgon J. Electrophoretic transfer of

proteins from polyacrylamide gels to nicoticellulose sheets:

procedure and some applications. Proc Natl Acad Sci USA

1979; 76: 4350-4·.

28. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein

measurement with pholin phenol reagent. J Biol Chern

1951;193:265-75.

24

29. Wiswedel I, Trumper L, Schid L, Augustin W. Injury of

mitochondrial respiration and membrane potential during

iron/ascorbate-induced peroxidation. Biochim Biophys Acta

1988;934:80-6.

30. Rouslin W. Mitochondrial complexes I, II, III, IV, and V 1n

myocardial ischemic and autolysis. Am J Physiol

1983;244:H743-8.

31. Dupuis A, Vignais PV. Interaction between the oligomycin

sensitivity conferring protein and the Fa sector of the

mitochondrial .adenosinetriphosphatase complex: Cooperative

effect of the F1 sector. Biochemistry 1987;26:410-8.

32. Penefsky HS. Mechanism of inhibition of mitochondrial

adenosine triphosphatase by dicyclohexylcarbodiimide and

oligomycin: Relationship to ATP synthesis. Proc Natl Acad Sci

USA 1985;82:1589-93.

33. Citron NM, Pedersen PL. A protein inhibitor of the

mitochondrial adenosine triphosphatase complex of rat liver -

purification and characterization. J Biol Chem 1979;254:3439-

43.

34. Gometz-Fernande.s JC, Harris DA. A thermodynamic analysis of

25

the interaction between the mitochondrial coupling adenosine

triphosphatase and its naturally occurring inhibitor protein.

Biochem J 1978;176:967-75.

35. Child P, Rafter J . Calcium enhances the hemolytic action of

bile salts. Biochim Biophys Acta 1986;855:357-64.

36. Billington D, Evans CE, Godfrey PP, Coleman R. Effects of bile

salts on the pllasma membranes of isolated rat hepatocytes.

Biochem J 1980;; 188:321-27.

37. Mcloughlin OJ, Howell ML, Bilirubin inhibition of enzymes

involved in the mitochondrial malate-aspartate shuttle.

Biochim Biophys. Acta 1987; 893: 7-12.

38. Goeser T, Nakata R, Braly LF, et al. The rat hepatocyte plasma

membrane organic anion binding protein is immunologically

related to the mitochondrial F1 adenosine triphosphatase beta

subunit. J Clin Invest 1990;86:220-7.

39. Mustafa MG, Cow:ger ML, King TE. Effects of bi l i.rubin on

mitochondrial r1eactions. J Biol Chern 1969;244:6403-14.

40. Wennberg RP. The importance of free bilirubin acid salt 1n

bilirubin uptake by erythrocytes and mitochondria. Pediatr Res

1988;23:443-7.

26

Table I.

Changes in respirator)l control ratio, ADP/0 ratio, state 3 respiration, and

phosphorylation rate c1fter induction of hemorrhagic shock in control and

jaundiced rats.

Glutamate as substrate

RC State 3 ADP/0 (nO/min per mg)

PR (nATP/min per mg)

-----------------------------------------------------------------Control (6) 5.72±0.07 44. 2±1.8 2.77±0 .04 122.1±3.7 (Sham)

After induction of hemorrhagic shock

1hr (8) 5.97±0.3.3 44.8±1.2 2. 72±0.03 121.6±2. 5

2hr (6) 5.76±0.22 44.1±1.6 2.76±0.03 121.5±;4.0

Jaundiced (6) 5.86±0.26 44 .5±2.6 2.87±0.04 127.3±2.3 (CBL)


1hr (8) 4. 85±0. 1(~0 37 .6±1.2b 2.77±0 .04 104.1±2.6c

2hr (6) 2.98:t0.1Sc 26. 5±1. 5b 2.29±0 .060 60.4±3.1C

(continued)

27

Succinate as substrate

RC State 3 (nO/min per mg)

ADP/0 PR (nATP/min per mg) _____________________________________________________________ , _____

Control (6) 5.16±'~. 22 95.0±2.8 1.84±0.07 173 .9±5.4 (Sham)


1hr (8) 5.05~3.12 98.4±2.4 1.83~.04 179.9±5.9

2hr (6) 4.81±(~.12 96 .6±3.9 1.741;0.03 167.6±6.9

Jaundiced (6) 4.80±_0.08 96.7±3.3 1. 79±_0.04 172.8±12.7 (CBL)


1hr (8) 3. 73~ .18C 82.8±3.7b 1. 741;0.06 142.7±4.3c

2hr (6) 2.36±0 . 10C 67.1±5.6b 1.46±0.130 95.9±5.8C

Values are means ± standard error of mean; number of rats is shown in

parentheses.

The abbreviations used are: RC = respiratory control ratio; State

3 = state 3 respiration (n mole of atom oxygen per min per mg of

mitochondrial protein); ADP/0 = mole of ATP synthesized per mole of atom

oxygen consumed; PR = phosphorylation rate (n mole of ATP synthesized per min

per mg of mitochondriQl protein)

Sham= sham operation, CBL =common bile duct ligation.

a P<0.05 compared with control groups

b P<0.01 compared with control groups

c P<0 .001 compared with control groups

28

Table II.

Changes in respirati<>n rate of liver mitochondria in the presence of FCCP

after induction of hemorrhagic shock in control and jaundiced rats. ----

Oxidative activity of liver mi tochondria

Substrate Glutamate (nO/min per mg)

Succinate (nO/min per mg)

Control (6) (Sham)

53.4±1. 7 144.7±5.9


lhr (8)

2hr (6)

55.2±2.6

52.4±3.7

Jaundiced (6) 54.6±6.6 (CBL)

After induction of hemorrhagi c shock

lhr (8) 52.2±2 .6

2hr (6) 45.4±2.3

144. 7±5.0

138 .9±8.2

138.4±1.4

136.7±3. 7

139.6±5.8


parentheses. The abbreviations used are: Sham = sham ope ration, CBL = common

bile duct ligation.

29

Table III.

Changes in membrane potential of liver mitochondria after induction of

hemorrhagic shock in control and jaundiced rats.

Control (Sham)

(4)


state 1 (mV)

124±5

1 hr (8) 126±5

2 hr (4)

Jaundiced (4) (CBL)


1 hr (6) 127±5

2 hr (4)

state 4 (mV)


parentheses.

The abbreviations used are: Sham = sham operation, CBL common bile duct

ligation.

30

Table I V.

Changes in ATPase activity after induction of hemorrhagic shock in control and

jaundiced rats

Control (6) (Sham)

After induction

Vmax (~mol/min per mg)

Km (mM ATP)

1.16±0 .02 0.20±0.05

of hemorrhagic shock

1hr

2hr

Jaundiced (CBL)

(8)

(6)

(6)

After induction of hemorrhagic

1hr (8)

2hr (6)

1.18±0.06

1.16±0.07

1.14±0.09

shock

0.611;0 .02a

0.32±0.05a

0.21±0.02

0.20±0.05

0.19±0.03

0.26±0.02b

0.27±0.04b


parentheses .

The abbreviations used are: Sham= sham operation, CBL =common bile duct

ligation

a P<0.001 compared with control groups.

b P<0.02 compared with control groups.

31

Table V.

H+-ATPase activity of liver mitochondria at 1 hr after induction of

hemorrhagic shock in control and jaundiced rats. (Oligomycin sensitivity and

affinity of F1 for the inner membrane)

Fractlona ATPase actl vlty

total unitsb oligomycin sens i tl vi ty

ESMP

Control 100±6 88±1

Jaundiced 52:t4C 67±1C

Supernatant from ESMP preparation

Control 4±1 78±3

Jaundiced 34±2c 16±1c

Values are means ± standard error of mean.

The abbreviation used is: ESMP = EDTA submitochondrial particle.

a Ten sham operated rats and jaundiced rats were utilized in this experiment.

b From 100 mg of mitochondria protein.

c P<0.001 compared with control groups.

32

Legend for figure~

Fig. 1. Western blot with anti-bovine heart mitochondrial F1

antibody. Lane a, molecular weight standards; lane b, purified

bovine heart mitochondrial F1 (0.2 ~g protein); lane c, ESMP

obtained from control rat liver at 1 hr after induction of

r hemorrhagic shock (10 ~g protein); lane d, ESMP obtained from

jaundiced rat liver at 1 hr after induction of hemorrhagic shock

(10 ~g protein). a : a subunit, ~: ~ subunit. The molecular mass

values for the standards are indicated on left in kilodalton. The

a and f3 subunits are the two prominent bands observed between the

66.2 and 45 kilodalton standards.

33

--a

.·

..

..

.. ..

1,·

. '.

, .

.. . ..

'·

.·

. ..

•.· . ,

I I

·.

.\

. . .. . \ ... , , ..

.. . ,.

. ....

. ~ • . t

;

' ...

.... • .

, . •, .

'I

l '·· • . ..

. .. . .

. •

肝機能障害の評価法とその障害機序に関する研究; ヒト肝 Title ... ·...

Documents

Transcript of 肝機能障害の評価法とその障害機序に関する研究; ヒト肝 Title ... ·...